Abstract

Alzheimer’s disease (AD) is the leading cause of dementia in the elderly. Incountries with aging populations, such as Australia, the prevalence of AD isprojected to increase substantially. AD is characterised by two distinctivepathological lesions in the brain, amyloid plaques and neurofibrillary tangles. Themajor component of amyloid plaques is an aggregating protein termed the betaamyloidprotein (Aβ). Aβ is formed normally from a larger precursor protein,known as the beta-amyloid precursor protein (APP). Although APP is centrallyinvolved in the pathogenesis of Alzheimer’s disease and the production of Aβ,relatively little is known about its normal function. Deciphering the function ofAPP in the brain may be essential for the development of effective ADtherapeutics.APP is a type I transmembrane glycoprotein that can be proteolytically processedby α, β- and γ-secretases to produce a number of secreted ectodomain fragmentstermed sAPPβ, sAPPα, Aβ and p3. Many studies have suggested that sAPPα mayact in the maintenance and development of the central nervous system, by actingas a paracrine factor. In vitro, sAPPα has been reported to modulate theproliferation and differentiation of a variety of cell types. However, themechanistic basis for these effects is unclear. In part, this uncertainty has arisenbecause the cell-surface receptor molecules that interact with sAPPα are notknown.Previous studies have reported that sAPPα may interact with a novel lipid-rafttype membrane domain in the cell. Furthermore, sAPPα has been reported to bindto the lipid GM1-ganglioside. On the basis of these reports, the work in this thesisexplored the hypothesis that an interaction of APP with cell surface lipids couldfacilitate binding and/or signalling by sAPPα. To determine if sAPPα is able to interact with a sub-group of lipids. The relativeability of sAPPα to bind to 27 physiological lipids was examined using a proteinlipidoverlay assay. This assay identified that sAPPα could bind selectively tophosphoinositide lipids (PIPs). Further, a recombinant fragment of APPcorresponding to the E1 N-terminal domain (APP-E1) also bound selectively toPIPs, suggesting there is a PIP-binding region within the E1 domain of APP.To investigate whether APP and PIP could interact on the cell surface, it was firstnecessary to demonstrate that PIPs are present on the cell surface. A live cellimmunolabelling method was used to examine the location of cell surface PIPs.Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) immunoreactivity was found tobe present on the surface of cells in primary murine hippocampal cultures indiscrete puncta <1 μm in size. This observation was also confirmed using arecombinant PI(4,5)P2 biosensor protein.To examine whether APP could interact with cell-surface PIP, studies wereperformed to examine the degree of colocalisation of exogenous APP-E1 and cellsurfacePI(4,5)P2. APP-E1 that was added to primary hippocampal cultures boundto the surface of neurons in discrete puncta <1 μm in size. The cell-bound APPE1and the cell-surface PI(4,5)P2 were highly co-localised on the surface ofneurons. However, cell-surface PI(4,5)P2 was also present on glial cells in culturewhere APP-E1 did not bind. Furthermore the binding of APP-E1 to cells couldnot be inhibited using a water soluble analogue of PI(4,5)P2. Therefore, these datasuggested that APP-E1 interacts with cell-surface PI(4,5)P2, but the interactionwas not sufficient to explain why APP-E1 binds to the cell surface.As the APP E1 domain contains a heparin-binding site, the role of this region wasinvestigated in the binding of APP-E1 to PIP and also the binding of APP-E1 tocells. Heparin did not block the binding of APP-E1 to PIP in vitro, suggesting theheparin-binding region and the PIP-binding region in the APP E1 domain are distinct. However, heparin did inhibit the binding of APP-E1 to cells, suggestingthat the heparin-binding region of APP is required for binding to cells.Furthermore, heparitinase treatment of cells significantly reduced cell surfaceheparan sulfate immunoreactivity, but did not affect the binding of APP-E1 tocells. These results suggest that APP may interact with PIP on the cell surfacealong with another cell surface component that binds to the heparin-binding site,which is not heparan sulfate.As PIPs are involved in many aspects of cellular physiology, it was hypothesizedthat APP may signal through modulation of levels of PIPs. To address thishypothesis, levels of PIPs were measured in primary cortical cultures by twomethods. Firstly, a mass-spectroscopy based method was developed to measuretotal levels of cellular PIP. No change in total PIP levels upon sAPPα treatmentcould be detected using this method. Secondly, levels of cell-surface PIPs weredetermined using an array of anti-PIP biosensors and antibodies. Under restingconditions, only PI(4,5)P2 was present on the surface of cells. However, in thepresence of APP-E1, there was an increase in the level of cell surface PI(3,4,5)P3and an increase in the level of PI(4,5)P2, indicating that APP binding to cells mayresult in an increase level of cell surface PIPs.The data presented in this thesis demonstrate that APP has a novel N-terminalPIP-binding domain. This domain may play a role in the normal function of APP,by facilitating PIP-dependent signalling.